Ligation of the cell surface molecule CD44 by anti-CD44 monoclonal antibodies (mAbs) has been shown to induce cell differentiation, cell growth inhibition and in some cases, apoptosis in myeloid leukemic cells. We report, herein, that exposure of human erythroleukemic HEL cells to the anti-CD44 mAb A3D8 resulted in cell growth inhibition followed by caspase-independent apoptosis-like cell death. This process was associated with the disruption of mitochondrial membrane potential (DWm), the mitochondrial release of apoptosis-inducing factor (AIF), but not of cytochrome c, and the nuclear translocation of AIF. All these effects including cell death, loss of mitochondrial DWm and AIF release were blocked by pretreatment with the poly (ADP-ribose) polymerase inhibitor isoquinoline. A significant protection against cell death was also observed by using small interfering RNA for AIF. Moreover, we show that calpain protease was activated before the appearance of apoptosis, and that calpain inhibitors or transfection of calpain-siRNA decrease A3D8-induced cell death, and block AIF release. These data suggest that CD44 ligation triggers a novel caspaseindependent cell death pathway via calpain-dependent AIF release in erythroleukemic HEL cells.

We have recently reported that ligation of the CD44 cell surface antigen with A3D8 monoclonal antibody (mAb) triggers incomplete differentiation and apoptosis of the acute promyelocytic leukemia (APL)-derived NB4 cells. The present study characterizes the mechanisms underlying the apoptotic effect of A3D8 in NB4 cells. We show that A3D8 induces activation of both initiator caspase-8 and -9 and effector caspase-3 and -7 but only inhibition of caspase-3/7 and caspase-8 reduces A3D8-induced apoptosis. Moreover, A3D8 induces mitochondrial alterations (decrease in mitochondrial membrane potential DW m and cytochrome c release), which are reduced by caspase-8 inhibitor, suggesting that caspase-8 is primarily involved in A3D8-induced apoptosis of NB4 cells. However, the apoptotic process is independent of TNF-family death receptor signalling. Interestingly, the general serine protease inhibitor 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF) decreases A3D8-induced apoptosis and when combined with general caspase inhibitor displays an additive effect resulting in complete prevention of apoptosis. These results suggest that both caspase-dependent and serine protease-dependent pathways contribute to A3D8-induced apoptosis. Finally, A3D8 induces apoptosis in all-trans-retinoic acid-resistant NB4-derived cells and in APL primary blasts, characterizing the A3D8 anti-CD44 mAb as a novel class of apoptosis-inducing agent in APL.

Acute myeloid leukemia (AML) is a clonal malignant disease characterized by an increasing number of immature myeloid cells arrested at various stages of granulocytic and monocytic differentiation. The stage of the blockage defines distinct AML subtypes (AML1 to AML5 are the most frequent ones). There is increasing evidence that the malignant clone is maintained by rare AML stem cells endowed with self-renewal capacity, which through extensive proliferation coupled to partial differentiation, generate leukemic progenitors and blasts, of which the vast majority have limited proliferative capacity. Contrarily to chemotherapy alone, which is still unable to cure most AML patients, the differentiation therapy, which consists in releasing the differentiation blockage of leukemic blasts, has succeeded, when it is combined with chemotherapy, to greatly improve the survival of AML3 patients, using retinoic acid as differentiating agent. However, this molecule is ineffective in other AML subtypes, which are the most frequent. We have shown that specific monoclonal antibodies (mAbs, H90 and A3D8) directed to the CD44 cell surface antigen, that is strongly expressed on human AML blasts, are capable of triggering terminal differentiation of leukemic blasts in AML1 to AML5 subtypes. These results have raised the perspective of developing a CD44-targeted differentiation therapy in most AML cases. Interestingly, these anti-CD44 mAbs can also induce the differentiation of AML cell lines, inhibit their proliferation and, in some cases, induce their apoptotic death. These results suggest that H90 and/or A3D8 mAbs may be capable to inhibit the proliferation of leukemic progenitors, to promote the differentiation of the leukemic stem cells at the expense of their self-renewal, and, perhaps, to induce their apoptotic death, thereby contributing to decrease the size of the leukemic clone. The challenges of an anti-CD44 based differentiation therapy in AML, and its importance in relation to the new other therapies developed in this malignancy, are discussed in this review.